PROJECT SUMMARY/ABSTRACT The goal of this proposal is to develop a circuit-level understanding of GABAergic neurons in the lateral hypothalamic area (LHA) by resolving their molecular and functional diversity and local and long-range connectivity. The LHA is a linchpin in the orchestration of fundamental aspects of behavior including arousal, stress, feeding and motivated behavior. Owing to its unique position at the intersection of multiple neural and humoral systems, the LHA drives essential behavioral programs that maintain homeostatic balance in physiology and behavior. Underlying the diverse functions of the LHA is an exceptionally heterogeneous population of neuronal cell types, of which few have been identified. Inhibitory GABAergic neurons in the LHA (LHAGABA) have emerged as potent actuators of feeding, reward and stress. However, within this broad class of neurons lie poorly resolved but functionally important subpopulations, which may differentially determine behavioral output. Conventional methods are limited in their ability to decipher the molecular and functional diversity of LHAGABA neurons, which represents a major barrier in our understanding of the role of LHA circuits in health and disease. There is, therefore, a critical need to systematically delineate the neurochemical identity, cellular properties and connectivity of functional subpopulations of LHAGABA neurons as a prerequisite to circuit-level behavioral analyses. To fill this knowledge gap, we will establish the first large-scale and systematic molecular, cellular and connectivity analysis of LHAGABA neurons at single-cell resolution using an integrated approach of electrophysiological phenotyping, optogenetics-based circuit mapping and single-cell transcriptional profiling. Our central hypothesis is that LHAGABA neurons can be classified into distinct functional subtypes according to key molecular markers, electrical signatures and patterns of synaptic connectivity. We will probe this hypothesis in three Specific Aims. Aim 1 will systematically define the electrophysiological and neuromodulatory phenotypes of candidate LHAGABA neuron populations and refine these characteristics based on long-range projections to the locus coeruleus (LC). Aim 2 will determine the synaptic connectivity between populations of LHAGABA neurons and key, arousal-related targets, both within the LHA and the LC, using patch-clamp electrophysiology and optogenetics. Aim 3 will apply cutting-edge quantitative single-cell transcriptional profiling methods to decipher novel, molecularly distinct populations of LHAGABA neurons. Impact: As the first comprehensive neurochemical, electrophysiological and anatomical census of inhibitory cell types in the LHA, this project will yield functional subpopulations of LHAGABA neurons that, in turn, will allow the identification of key markers for the design of novel cre driver lines and intersectional genetic tools for circuit-specific dissection of LHAGABA circuits in behavior. Furthermore, a deeper understanding of LHAGABA cell types and circuits holds the promise of identifying therapeutic targets for neuropsychiatric illnesses, addiction, post-traumatic stress disorder, sleep and metabolic disorders.